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Exploring the interactome of the bacterial Sec translocon

University of British Columbia

Many periplasmic and extracellular Escherichia coli (E. coli) proteins are transported across the inner bacterial membrane through the highly conserved heterotrimeric SecYEG protein-conducting channel. During post-translational translocation, polypeptide substrates are driven across the membrane through SecYEG by the ATPase SecA, which binds to SecYEG and couples nucleotide hydrolysis to polypeptide movement. In the first part of this thesis, we study the dynamics of SecYEG-SecA interactions. We show that SecA is a highly dynamic enzyme, repeatedly binding to and dissociating from SecYEG during substrate translocation. Using two model Sec-dependent protein substrates, we show that the importance of these dynamics for efficient translocation depends on the length of the translocating protein substrate. In the second part of this thesis, we turn to quantitative proteomics to identify novel interactors of the SecYEG complex. Previous studies have identified and validated a series of membrane embedded interactors of SecYEG using classical detergent-based methods. However, it is possible that other important interactors of the Sec translocon may exist which have not yet been identified by detergent-based proteomic methods - the difficulties of using detergent-based methods to identify and characterize transient interactors of membrane proteins and complexes are well-documented. Here, we employ the peptidisc - a "one-size-fits-all" membrane mimetic - to identify and characterize potentially novel interactors of the Sec translocon in detergent-free conditions. One of the most notable interactions identified in this work is a super-complex between the Sec translocon and the outer membrane embedded Bam complex, which is required for insertion of outer membrane proteins (OMPs). This observation is particularly astonishing and has implications for our understanding of outer membrane protein biogenesis. Finally, we develop a functionalized variant of the peptidisc scaffold and demonstrate its utility for isolation of the membrane proteome. As a simple case study, we employ the functionalized peptidisc scaffold to survey changes in the membrane proteome caused by altered gene expression. Potential future applications of the peptidisc membrane mimetic in the fields of membrane protein biochemistry and membrane proteomics will also be discussed.

Text

2024-04-30

Attribution-NonCommercial-NoDerivatives 4.0 International

http://hdl.handle.net/2429/74194

Biochemistry and Molecular Biology

University of British Columbia

UBCV

http://creativecommons.org/licenses/by-nc-nd/4.0/